U.S. Pat. No. 4,886,750 discloses the use of esterases in the stereoselective hydrolysis of esters of 2-arylpropionic acids. In this document the enzyme responsible for the hydrolysis of (S)-naproxen esters is characterized. The corresponding esterase gene was obtained from the Bacillus subtilis Thai 1-8 strain (CBS 679.85). This gene encoding the enzyme responsible for the stereoselective conversion of (R,S)-naproxen ester was cloned in E. coli and Bacillus subtilis. It was found that the esterase activity was improved by introducing multiple gene copies in several Bacillus subtilis (a.o. CBS 673.86). The suitability of the microorganism and the enzyme derived therefrom for use in a process to hydrolyse S-naproxen ester was therefore also improved.
In said U.S. patent only low substrate concentrations (naproxen or ibuprofen) are used. In contrast, commercial applications require high product concentrations in order to obtain economically attractive results. However, during tests at high substrate concentrations (commercial conditions) irreversible inactivation of the enzyme has been noticed. For example, carboxyl esterase obtained from Bacillus Thai I-8 was almost completely inactivated within one hour when 30 g/l naproxen ester was added (pH=9, T=40.degree. C. and Tween 80 (TM) medium). The esterase as such is stable at pH=9 and T=40.degree. C. (with and without Tween 80 (TM)) for several hours. During the stereoselective hydrolysis of (R,S)-naproxen ester, the enzyme was inactivated by the naproxen formed during the hydrolysis. High yields of naproxen could not therefore be obtained.
The enzyme carboxyl esterase may be used in several other stereospecific esterase hydrolysis reactions. However, it is found that the product (the acid) of these reactions often inactivates the enzyme when the reaction takes place at commercially interesting starting concentrations of the ester.
The carboxyl esterase can be used in the stereospecific hydrolysis of diclofop esters, resulting in the corresponding enantiomeric pure (S)-acid, which process is described in EP-A-0299559. The diclofop formed will inactivate the enzyme under commercially attractive conversion conditions.
Other compounds that inactivate the enzyme are, for example, 2-naphthoxy acetic acid, ibuprofen, 2-naphthol and phenol.
In the literature enzymes are known to become inactivated because of their low thermal stability. At elevated temperatures unfolding of the enzyme may take place. Heat treatment causes especially the hydrogen bonds to break (see e.g. R. D. Schmid, Advances in Biochemical Engineering 12, Ghose, Fiechler & Blakebrough (Eds), Springer, Berlin (1979) pp. 41-115). Thermo unfolding of enzymes can, however, be diminished by immobilization or cross-linking of the enzyme. For example, cross-linking with glutaraldehyde improved the thermostability of Papain (Royer et al., FEMS Lett. 80 (1977) 1) and Subtilopeptidase (Boudrant et al., Biotechnol. Bioeng. 18 (1976) 1719).
Even the mechanism of thermostabilisation is not well understood. E. T. Reese and M. Manders (Biotechnol. Bioeng. 22 (2) 1980 pp. 326-336 showed that cross-linking (glutaraldehyde treatment) did not result in an increase of thermostability and activity of cellulase. Similar results were found by N. W. Ugarova (Biokhimiya 42 (7), 1977 pp. 1212-1220) who reported that modification of peroxidase with glutaraldehyde gave a 2.5-fold decrease in thermostability.
The prior art presents only very specific solutions for specific problems (immobilization and cross-linking techniques) which are not generally applicable. Moreover it has been noticed that the carboxyl esterase is not thermally inactivated at normal reaction conditions (up to 45.degree. C.) but is only inactivated by certain compounds at reaction conditions. The prior art is silent on such kind of inactivations.
When the amino acid residue which is the cause of inactivation of the protein is known, an alternative approach to chemical modification is available. In that case one can replace the residue for another one by site-directed mutagenesis, as described for instance by Ausubel et al. (Current Protocols in Molecular Biology, John Wiley & Son Inc., 1987, New York). In this way e.g. the oxidation resistance of B. alcalophilus serine protease was improved by replacing a methionine residue by a serine residue (European patent application 0328229).
The known stabilization techniques cannot be applied as such to the present enzyme because the nature of the inactivation is different when inactivation by chemical compounds plays a role.